Our laboratory investigations are focused on studying genes and pathways that regulate growth and programmed cell death of epithelial cancer cells with a focus on breast and ovarian cancer. These investigations will be used as the basis for preclinical development of targeted therapeutics to breast and ovarian cancers including the characterization of the mechanisms that regulate sensitivity and resistance of cancer cells to targeted therapeutics. Ongoing projects include: (1) the regulation of growth factor receptor function by cbl proteins; and (2) the function of death receptors in epithelial cancer cells. (1) The regulation of growth factor receptor function by cbl proteins. The Epidermal Growth Factor Receptor (EGFR) family (which includes the EGFR, erb-B2, erb-B3, and erb-B4) is a family of growth factor receptors which are frequently activated by amplification and mutation in epithelial malignancies including breast cancer. Understanding the mechanisms that regulate signaling by these receptors should uncover new ways to inhibit cancer cell growth. Genetic studies in the nematode, C. elegans, suggest that the cbl proteins are inhibitors of EGFR signaling. We have cloned cbl-b and cbl-3, two new mammalian genes with homology to the c-cbl proto-oncogene. Our work is focused on elucidating the role of cbl proteins in regulating the function of the EGF family of receptor tyrosine kinases in mammary epithelial cells. We have demonstrated that cbl-b and cbl-3 inhibit the function of the activated EGFR. cbl-b and cbl-3 become phosphorylated and associate with the EGF receptor upon activation and inhibit multiple downstream signaling pathways. In collaboration with Yossi Yarden at the Weizmann Institute of Science, we have shown that cbl proteins act as E3 ligases for the activated EGFR and that this results in ubiquitination and degradation of the activated EGFR (but not of the inactive EGFR). Therefore, cbl proteins negatively regulate EGFR function by degrading the active receptors. Ongoing biochemical work is exploring the structure-function relationships necessary for cbl proteins to function as E3 ligases and the differences in the specificity and/or function of the three mammalian cbl proteins. We are also studying the role of the different cbl proteins in vivo using cbl-b and cbl-3 null mice. (2) The function of death receptors in epithelial cancer cells. Cancer cells avoid apoptosis by a variety of genetic and epigenetic mechanisms. We are investigating the expression and function of death receptors of the TNFR family (e.g. TNFR, Fas, Dr3, Dr4, and Dr5) and their ligands (e.g. TNF, Fas Ligand, and TRAIL) in normal and malignant breast and ovarian epithelial cells in order to selectively trigger apoptosis in the cancer cells. In our initial studies of the Fas receptor, we demonstrated that most breast cancer cells are resistant to Fas-induced apoptosis while the normal breast epithelial cells are sensitive. Treatment of resistant breast cancer cells with interferon-g increased the expression of the Fas receptor and of downstream caspases. This reversed the resistance of breast cancer cells to Fas-induced apoptosis. We have shown that most normal and malignant breast cancer cell lines are resistant to the induction of apoptosis by TRAIL, the ligand for Fas-related death receptors DR4 and DR5. This resistance can be overcome by co-incubation of the cells with sub-toxic doses of drugs such as adriamycin. More recently, we have shown that many ovarian cancer cell lines are also resistant to TRAIL-induced apoptosis and again that chemotherapeutic agents, commonly used to treat ovarian cancer, synergistically enhance TRAIL-induced apoptosis. We have also shown that trastuzumab, the monoclonal antibody to the erbB2 receptor, specifically enhanced TRAIL-induced apoptosis in cancer cells which overexpress the erbB2 receptor but not in cells with normal or low levels of erbB2. Thus, this may allow us to target TRAIL-induced apoptosis to a subset of cancer cells without affecting normal cells. Mechanistically, we have shown that trastuzumab enhances TRAIL-induced apoptosis by downregulating the erbB2 receptor and that this results in decreased AKT activity. Our current work is focused on understanding the mechanisms that underlie the resistance to death mediated by TRAIL ligand in breast and ovarian cancer cells. Our work includes screening death receptor pathway components for evidence of mutations that could account for the resistance of tumor cells to apoptosis. In addition, we are investigating mechanisms to overcome this resistance.